Method of inducing moult of silkworms



United States Patent 2 Claims. ((31. 119-6) The present application is adivision of copendin-g application, Serial No. 343,106, filed February6, 1964.

The present invention relates to including moult of silkworms, and moreespecially relates to a method of inducing moult of silkworms whenbreeding the latter, by administering to the silkworms an artificialdiet containing at least one of the following choline derivatives:

Choline chloride, Acetylcholine chloride, Choline .phosphochloride,Lecithin,

Benzoylcholine chloride, or A mixture of these derivatives,

in an amount of not less than 500 gamma-of dried artificial diet.

While silkworms are known to be monophargous in-- eating nothing butmulberry leaves, no exact reason for it has been made clear. Dr.Hamamura,- one of the inventors of this invention, found out that inmulberry v leaves there are contained such factors as the attractingfactor which attracts silkworms (terpenes such as citral, linalylacetate, linalol, terpinyl acetate), the biting factor which stimulatessilkworms to bite (B-sitosterol with or without flavonoids such asquercetin, morin, rutin, isoquercitrin), the swallowing factor whichstimulates them to bite and swallow continuously (cellulose powder), andsugar such as sucrose, fructose, glucose and inorganic phosphate such aspotassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodiumdihydrogen phosphate, disodium hydrogen phosphate, calcium phosphate,and these are indispensable for the feed of silkworms. Conditions of thefoodstutf, e.g. taste and nutritional value, are inferred strictly to beof merely secondary significance in inducing silkworms to the action ofeating, though they should certainly be considered in connection withthe insectal growth.

The present inventors have extensively studied on the feeding ofsilkworms so as to rear silkworms with artificial diet without anyaddition of mulberry leaves, and found out that when newly hatchedsilkworms are reared with diet prepared by replacing defatted soybeanpowder in the basal artificial diet employed in Example 1 shown laterwith pure soybean protein, molting is suppressed and no larva reachesthe 2nd instar. Accordingly the present inventors have named thesubstance which is contained in defatted soybean powder but not in puresoybean protein as the growth-promoting factor. Further con- 3,230,930Patented Jan. 25, 1966 tinuous study has made it clear that thegrowth-promoting factor is choline or its derivaties. Namely, it hasbeen found out by the present inventors that choline or its derivativeshave a distinguishable action on inducing moult of silkworms. In thepresent specification, choline means choline salts. As choline salts,choline halides are most conveniently put to use. Among the cholinederivatives, there may, for example, be enumerated acetylcholine halide,lecithin, benzoylcholine halide, and choline phosphohalide. As thehalides, most preferable are chlorides.

The first object of this invention is to breed silkworms with anartificial diet. The second object is to promote the growth ofsilkworms. The first object is realized by preparing foodstuffcontaining choline or its derivatives. The second and third objects arerealized by giving silkworms foodstuif containing choline or itsderivatives.

The growth-promoting factor of this invention shows especially strongeffect when given at a period from larvae to the 2nd instar.

For a practical application choline derivatives may preferably beutilized together with the afore-mentioned attracting factor, bitingfactor and swallowing factor, i.e. terpenes, fl-sitosterol with orwithout flavonoids and cellulose powder, though terpenes may be omittedwhen silkworms are put on the feed, and sugar, inorganic phosphate,inorganic silicate, inositol, etc. by mixing various componentsnecessary for the growth of silkworms, in a powdery, jelly or pasteform. As the components which are necessary for the growth of silkworms,there may, for example, be agar-agar, sodium carboxymethyl cellulose,methylcellulose, alginic acid, soybean powder, parched bean flour,defatted soybean powder, bean curd, dried fermented soybean powder,defatted and dried bean paste; cereal flour such as rice flour, barleyflour, wheat flour, or their processed matter; yeast or its processedmatter such as dry yeast, yeast extract, extracted fluid of yeast,extracted cake of yeast; straw ashes; fish meal;

Animal or plant protein;

Amino acid such as glutamic acid, aspartic acid, leucine, lysine,tyrosine, tryptophane, valine, serine, proline, glycine, alanine,isoleucine, phenylalanine, arginine, methionine, threonine or theirsalts;

Vitamins such as pantothenic acid, nicotinic acid, vitamin B vitamin Bvitamin B vitamin B vitamin C, vitamin D, biotin, folic acid, vitamin K,vitamin E, vitamin P, inositol, orotic :acid, lipoic acid;

Inorganic salts such as calcium carbonate, sodium carbonate, sodiumhydrogen carbonate, sodium chloride;

Honey collected from beehive or its processed matter;

Pectin;

Enzyme such as diastase, papain, trypsin;

Preservative such as formalin, sodium dehydroacetate, vitamin K sodiumsorbate;

Antibiotic such as tetracycline, chlortetracycline, oxytetracycline,streptomycin, dihydrostrep-tomycin, neornycin, kanamycin, picromycin,leucomycin, erythromycin, oleandomycin, trichomycin, chloramphenicol,penicillin;

Sulfa drug such as sulfaisomidine, sulfaisoxazole, methoxypyridazine;

Furan derivatives such as nitrofuran; and

Glucuronic acid or its salt such as sodium salt, potassium salt, calciumsalt, ammonium salt, organic amine salt, glucuronic acid ethyl ester,glucuronic acid amide, 2-amino-2-desoxy-D-glucuronic acid or its salt,N-gl'ucuronosylglucosamine or its salt.

The quantities of choline derivatives necessary for in ducing moult ofsilkworm-s may be varied depending on various conditions such as instaror the state of the silkworms :and the kind or quantity of the othercomponents composing the artificial diet. Generally, it is necessary toemploy not less than 500 gamma per one gram of the dried artificialdiet. Generally an amount of choline derivatives over 500 gamma per 1gram gives the same result as in 500 gamma/ one gram.

Morin, milligrams 25 Inositol, milligrams 50 K HPO milligrams 50 SiOmilligrams 250 5 Water, cubic centimeters Table 2.Dl'et Table 3.ResultsThe following experiments are given to show the eflicacy for inducingmoult of silkworms by choline derivatives.

EXPERIMENT 1 Larvae of silkworms are reared with the basal artificialdiet I, whose components are shown in Table 1. And diets II, III and IVare prepared by changing components of the basal artificial diet. Whenlarvae of silkworms are reared with the basal artificial diet, they canreach the 2nd EXPERIMENT 2 Experiment is carried out in the same way asin Experiment 1 by employing diet V and VI shown in Table 4.

Table 4.Diet

Diet V.Basal artificial diet-l-dihydrostreptomycin, 10

milligrams. Diet VI.Diet V+acetylcholine chloride, 5 milligrams.

Table 5.Results Number of silkworms Number of Weight of Number ofreached the 2nd instar silkworms each silkworm Diet larvae of silkwhichsurvived of the 2nd inworms emup to the 2nd star (milliployed for the 67 8 9 instar grams) test days days days days I 20 2 2 6 2 12 4. 5 V 20 57 12 .5. 4 VI 2O 10 7 17 5. 4

instar. While, when larvae of silkworms are reared with the diet IIwhich is prepared by replacing defatted soybean powder in the basalartificial diet with pure soybean protein, molting is suppressed and nolarva reaches the 2nd instar. Further, when larvae of silkworms arereared with the diet III or IV which is prepared by adding acetylcholinerespectively to the basal artificial diet and to the diet II, larvae canmolt to be the 2nd instar, and time required for silkworms to reach the2nd instar is shortened.

Table 1.-The components of basal artificial diet Cellulose powder, grams5.0 Defatted soybean powder, grams 2.0 Starch, grams 1.5 Sugar, gram 1.0Wessons minerals, gram 0.090 Vitamins:

Vitamin C, gram 0.04 Vitamin B milligram 0.01 Vitamin B milligram 0.01Vitamin B milligram 0.01 Nicotinic acid, milligram 0.02 Pantothenic acid(calcium salt), milligram 0.02 Folic acid, milligram 0.002 Biotin,milligram 0.002 Vitamin B milligram 0.01 fi-Sitosterol, milligramsEXAMPLE Foodstuff for silkworms containing 2.5 parts by weight ofcellulose powder, 1.5 parts by weight of defatted soybean protein, 2.0parts by weight of frozen mulberry leaves powder, 0.5 part by weight ofyeast, 1.5 parts by weight of starch, 1.0 part 'by weight of sugar,0.015 part by weight of dihydrostreptomycin, 0.005 part by weight ofacetylcholine chloride, and a small amount of fi-sitoisterol, Wessonsminerals, morin, inositol, K HPO and SiO and 15 parts by volume ofwater.

Having thus disclosed this invention, what is claimed is:

'1. A method for inducing moult of silkworms in breeding with anartificial diet, which comprises feeding silkworms, in a moulting stagethereof, a member selected from the group consisting of cholinechloride, acetylcholine chloride, choline phosphochloride, lecithin,benzoylcholine chloride .and a mixture thereof, in an amount of not lessthan 500 'y/ g. of dried artificial diet.

2. A method for inducing moult of silkworms in breeding, With anartificial diet, which comprises feeding silkworms, in a moulting stagethereof, acetylcholine chloride in an amount of not less than 500 gammaper one gram Hamamura et a1.: Food Selection by Silkworm Larvae,

of dried artificial diet. Nature, vol. 194, May 1962, pp. 754755.

Merck and Co., Inc., Merck Index, 7th edition (1960),

References Cited by the Examiner paper 2 5 3 UNITED STATES PATENTS 5Fukuda et 211.: Synthetic Diet for Silkworm Raising,

1990343 2/1935 Naruse Nature, vol. 196, October 1962, pp. 5354.

OTHER REFERENCES SAMUEL KOREN, Primary Examiner.

Rose et al.: Condensed Chemical Dictionary, 5th ed., ALDRICH F. MEDBERY,Examiner. page 11 (1956). 10

2. A METHOD FOR INDUCING MOULT OF SILKWORMS IN BREEDING WITH ANARTIFICIAL DIET, WHICH COMPRISES FEEDING SILKWORMS, IN A MOULTING STAGETHEREOF, ACETYLCHOLINE CHLORIDE IN AN AMOUNT OF NOT LESS THAN 500 GAMMAPER ONE GRAM OF DRIED ARTIFICIAL DIET.